U.S. patent number 9,315,810 [Application Number 14/123,562] was granted by the patent office on 2016-04-19 for oligonucleotide derivative, oligonucleotide derivative-containing pharmaceutical composition for treatment and pharmaceutical composition for diagnosis, and oligonucleotide derivative for regulation of mirna function.
This patent grant is currently assigned to National University Corporation Hokkaido University. The grantee listed for this patent is Akira Matsuda, Mayumi Takahashi. Invention is credited to Akira Matsuda, Mayumi Takahashi.
United States Patent |
9,315,810 |
Matsuda , et al. |
April 19, 2016 |
Oligonucleotide derivative, oligonucleotide derivative-containing
pharmaceutical composition for treatment and pharmaceutical
composition for diagnosis, and oligonucleotide derivative for
regulation of miRNA function
Abstract
An oligonucleotide derivative comprises repeating structural
units represented by the following general formula (wherein B
represents adenine, guanine, cytosine, or uracil; X represents a
sulfur atom or an oxygen atom; n represents an integer of 6 to 60;
and B and X are independently represented in each of the repeating
structural units), wherein X is a sulfur atom in at least one of
the repeating structural units represented by the general formula:
##STR00001##
Inventors: |
Matsuda; Akira (Hokkaido,
JP), Takahashi; Mayumi (Hokkaido, JP) |
Applicant: |
Name |
City |
State |
Country |
Type |
Matsuda; Akira
Takahashi; Mayumi |
Hokkaido
Hokkaido |
N/A
N/A |
JP
JP |
|
|
Assignee: |
National University Corporation
Hokkaido University (Hokkaido, JP)
|
Family
ID: |
47259467 |
Appl.
No.: |
14/123,562 |
Filed: |
June 1, 2012 |
PCT
Filed: |
June 01, 2012 |
PCT No.: |
PCT/JP2012/064276 |
371(c)(1),(2),(4) Date: |
December 03, 2013 |
PCT
Pub. No.: |
WO2012/165616 |
PCT
Pub. Date: |
December 06, 2012 |
Prior Publication Data
|
|
|
|
Document
Identifier |
Publication Date |
|
US 20140128347 A1 |
May 8, 2014 |
|
Foreign Application Priority Data
|
|
|
|
|
Jun 3, 2011 [JP] |
|
|
2011-125734 |
|
Current U.S.
Class: |
1/1 |
Current CPC
Class: |
C12N
15/113 (20130101); A61P 35/00 (20180101); A61P
3/00 (20180101); A61K 31/713 (20130101); A61K
31/7088 (20130101); A61P 7/00 (20180101); C12N
2310/315 (20130101); C12N 2310/351 (20130101); C12N
2310/113 (20130101); C12N 2310/3235 (20130101); C12N
2310/321 (20130101); C12N 2310/3521 (20130101) |
Current International
Class: |
C12N
15/113 (20100101); A61K 31/7088 (20060101); A61K
31/713 (20060101) |
References Cited
[Referenced By]
U.S. Patent Documents
Other References
Bonci "MicroRNA-21 as Therapeutic Target in Cancer and
Cardiovascular Disease." Cardiovascular Drug Discovery, 2010, 5,
156-161. cited by applicant .
Broderick et al. "MicroRNA Therapeutics." Gene Therapy (2011) 18,
1104-1110. cited by applicant .
Cummins et al. "Characterization of fully 2'-modified
oligoribonucleotide hetero and homoduplex hybridization and
nuclease sensitivity." Nucleic Acids Research, 1995, vol. 23, No.
11 2019-2024. cited by applicant .
Dande et al. "Improving RNA Interference in Mammalian Cells by
4'-Thio-Modified Small Interfering RNA (siRNA): Effect on siRNA
Activity and Nuclease Stability When Used in Combination with
2'-O-Alkyl Modifications." J. Med. Chem. 2006, 49, 1624-1634. cited
by applicant .
Hoshika et al. "Synthesis and physical and physiological properties
of 4'-thioRNA: application to post-modification of RNA aptamer
toward NF-kB." Nucleic Acids Research, 2004, vol. 32, No. 13
3815-3825. cited by applicant .
Judge et al. "Design of Noninflammatory Synthetic siRNA Mediating
Potent Gene Silencing in Vivo." Molecular Therapy vol. 13, No. 3,
Mar. 2006. cited by applicant .
Lanford et al. "Therapeutic Silencing of MicroRNA-122 in Primates
with Chronic Hepatitis C Virus Infection." Science 327, 198, 2010.
cited by applicant .
Morrisey "The magic and mystery of miR-21." The Journal of Clinical
Investigation, vol. 120, No. 11, Nov. 2010. cited by applicant
.
Musso et al. "Emerging Molecular Targets for the Treatment of
Nonalcoholic Fatty Liver Disease." Annu. Rev. Med. 2010, 61:375-92.
cited by applicant .
Shan et al. "Reciprocal Effects of Micro-RNA-122 on Expression of
Heme Oxygenase-1 and Hepatitis C Virus Genes in Human Hepatocytes."
Gastroenterology 2007; 133: 1166-1174. cited by applicant .
Takahashi et al. "Synthesis and characterization of
2'-modified-4'-thioRNA: a comprehensive comparison of nuclease
stability." Nucleic Acids Research, 2009, vol. 37, No. 4,
1353-1362. cited by applicant .
Takahashi et al. "Intracellular stability of
2'.sub.--OMe-4'-thioribonucleoside modified siRNA leads to
long-term RNAi effect." Nucleic Acids Research, 2012m vol. 40, No.
12, 5787-5793. cited by applicant.
|
Primary Examiner: Vivlemore; Tracy
Attorney, Agent or Firm: K&L Gates, LLP Cullman; Louis
Bergman; Michelle Glasky
Claims
The invention claimed is:
1. A method for treatment comprising administering an
oligonucleotide derivative comprising repeating structural units
represented by general formula: ##STR00021## wherein B represents
adenine, guanine, cytosine, or uracil; X represents a sulfur atom
in all of the repeating structural units represented by the general
formula; n represents an integer of 6 to 60; and B and X are
independently represented in each of the repeating structural
units, wherein the oliqonucleotide derivative comprises a sequence
complementary to an entire sequence or a partial sequence of a
miRNA.
2. The method according to claim 1, wherein at least one ligand is
bound to the 5' end, the 3' end, or both the 5' end and the 3' end
of the oligonucleotide derivative.
3. The method according to claim 1, wherein the oligonucleotide
derivative comprises a sequence complementary to the entire
sequence or a partial sequence of a miRNA.
4. The method according to claim 3, wherein the miRNA is
miRNA-21.
5. The method according to claim 3, wherein the miRNA is
miRNA-122.
6. A method for diagnosis comprising administering an
oligonucleotide derivative comprising repeating structural units
represented by general formula: ##STR00022## wherein B represents
adenine, guanine, cytosine, or uracil; X represents a sulfur atom
in all of the repeating structural units represented by the general
formula; n represents an integer of 6 to 60; and B and X are
independently represented in each of the repeating structural
units, wherein the oligonucleotide derivative comprises a sequence
complementary to an entire sequence or a partial sequence of a
miRNA.
7. A method for inhibiting miRNA function comprising administering
an oligonucleotide derivative comprising repeating structural units
represented by general formula: ##STR00023## wherein B represents
adenine, guanine, cytosine, or uracil; X represents a sulfur atom
in all of the repeating structural units represented by the general
formula; n represents an integer of 6 to 60; and B and X are
independently represented in each of the repeating structural
units, wherein the oliqonucleotide derivative comprises a sequence
complementary to an entire sequence or a partial sequence of a
miRNA.
Description
CROSS REFERENCE TO RELATED APPLICATIONS
This application is a national phase of PCT/JP2012/064276 filed on
Jun. 1, 2012 which claims priority to Japanese Patent Application
JP2011-125734 filed on Jun. 3, 2011, the entire disclosures of
which are incorporated by reference herein.
TECHNICAL FIELD
The present invention relates to an oligonucleotide derivative, a
pharmaceutical composition for treatment and a pharmaceutical
composition for diagnosis that include the oligonucleotide
derivative, as well as to an oligonucleotide derivative for
inhibiting miRNA function.
BACKGROUND ART
In recent years, due to the discovery of RNA interference (RNAi),
the advancement of transcriptome analysis methods, and the like,
research on RNA has been dramatically expanded. It has been shown
that RNA regulates many biologically important functions, and
associations between RNA and various diseases including cancer have
been pointed out.
Studies on cancer treatment by regulation of RNA function have been
vigorously conducted and research and development of RNA-targeting
pharmaceuticals have been widely carried out. Above all, RNA
regulation method using a nucleic acid is based on the Watson-Crick
type base pair formation and uses a nucleic acid having a sequence
specific to a target RNA. Such an RNA regulation method directly
regulates RNA, thereby allowing the adjustment of cellular
function.
In recent years, as an RNA regulation method using a nucleic acid,
methodologies such as antisense method and RNAi method have been
established. In addition, studies on nucleic acid medicines for
inhibiting the function of microRNA (hereinafter referred to as
miRNA) have been advanced (Non Patent Literature 1).
Nucleic acid medicines produced by application of RNA regulation
method using a nucleic acid have problems to be solved, such as (1)
the loss of effect due to decomposition by nuclease present inside
and outside cells, (2) thermal instability of a double-stranded
higher order structure, (3) targeting of cells and tissues, and (4)
occurrence of side effects due to natural immune response.
In order to overcome these problems, in the creation of nucleic
acid medicines, studies have been conducted to provide functions
such as resistance against nuclease, thermal stability of the
double-stranded higher order structure, and an ability to avoid
natural immune response by performing some chemical modification on
a nucleic acid. It has been reported that 2'-O-methyl RNAs obtained
by the methylation of a hydroxyl group at position 2' of the sugar
moiety of a nucleic acid have nuclease resistance, thermal
stability, and the ability to avoid a natural immune response (Non
Patent Literature 2 and 3). In addition, it has been reported that
4'-thioRNA obtained by the substitution of an oxygen atom at
position 4' of a furanose ring of a nucleoside sugar moiety with a
sulfur atom has a nuclease resistance and thermal stability (Non
Patent Literature 4).
CITATION LIST
Non Patent Literature
Non Patent Literature 1: Robert E. Lanford et al., Science, 327,
198-201 (2010). Non Patent Literature 2: Commins L. L. et al.,
Nucleic Acids Res. 23, 2019-2024 (1995). Non Patent Literature 3:
Judge A. D. et al., Mol. Ther. 13, 494-505 (2006). Non Patent
Literature 4: Hoshika S., Minakawa N. and Matsuda A., Nucleic Acids
Res. 32, 3815-3825 (2004).
SUMMARY OF INVENTION
Technical Problem
However, the 2'-O-methyl RNAs described in Non Patent Literature 2
and 3 are not considered to be sufficiently stable in in vivo
environments and also are still problematic in terms of persistence
of effect. In addition, although the 4'-thioRNA described in Non
Patent Literature 4 has nuclease resistance, the development of
modified RNAs having more excellent nuclease resistance has been
desired in order to use in vivo as nucleic acid medicines.
The present invention has been accomplished in view of the above
circumstances, and the objectives of the present invention are to
provide an oligonucleotide derivative having excellent effect
persistence enough for in vivo use and thermal stability, a
pharmaceutical composition for treatment and a pharmaceutical
composition for diagnosis that include the oligonucleotide
derivative, as well as an oligonucleotide derivative for inhibiting
miRNA function.
Solution to Problem
In order to achieve the above objectives, an oligonucleotide
derivative according to a first aspect of the present invention
comprises repeating structural units represented by the following
general formula:
##STR00002## (wherein B represents adenine, guanine, cytosine, or
uracil; X represents a sulfur atom or an oxygen atom; n represents
an integer of 6 to 60; and B and X are independently represented in
each of the repeating structural units), wherein X represents a
sulfur atom in at least one of the repeating structural units
represented by the general formula.
X may represent a sulfur atom in all of the repeating structural
units represented by the general formula.
At least one ligand may be bound to the 5' end, the 3' end, or both
the 5' end and the 3' end of the oligonucleotide derivative.
The oligonucleotide derivative may comprise a sequence
complementary to the entire sequence or a partial sequence of a
miRNA.
The miRNA may be miRNA-21.
The miRNA may be miRNA-122.
A pharmaceutical composition for treatment according to a second
aspect of the present invention includes the oligonucleotide
derivative.
The pharmaceutical composition for treatment may inhibit miRNA
function.
A pharmaceutical composition for diagnosis according to a third
aspect of the present invention includes the oligonucleotide
derivative.
An oligonucleotide derivative for inhibiting miRNA function
according to a fourth aspect of the present invention comprises
repeating structural units represented by the following general
formula:
##STR00003## (wherein B represents adenine, guanine, cytosine, or
uracil; X represents a sulfur atom or an oxygen atom; n represents
an integer of 6 to 60; and B and X are independently represented in
each of the repeating structural units), wherein X is a sulfur atom
in at least one of the repeating structural units represented by
the general formula.
X may represent a sulfur atom in all of the repeating structural
units represented by the general formula.
At least one ligand may be bound to the 5' end, the 3' end, or the
5' end and the 3' end of the oligonucleotide derivative.
The oligonucleotide derivative may comprise a sequence
complementary to the entire sequence or a partial sequence of a
miRNA.
The miRNA may be miRNA-21.
The miRNA may be miRNA-122.
Advantageous Effects of Invention
The present invention can provide an oligonucleotide derivative
having excellent effect persistence enough for in vivo use and
thermal stability, a pharmaceutical composition for treatment and a
pharmaceutical composition for diagnosis that include the
oligonucleotide derivative, as well as an oligonucleotide
derivative for inhibiting miRNA function.
BRIEF DESCRIPTION OF DRAWINGS
FIG. 1 is a view depicting sequences and Tm values of individual
unconjugated AMOs 21;
FIG. 2 is a view depicting sequences and Tm values of individual
unconjugated AMOs 122;
FIG. 3 is a view illustrating a reporter vector used to evaluate an
inhibitory effect of each AMO on miRNA;
FIG. 4 is a view illustrating a protocol for evaluating the
inhibitory effect of each AMO on miRNA;
FIG. 5 is a graph depicting inhibitory effects of unconjugated AMOs
21 on miRNA-21;
FIG. 6 is a graph depicting inhibitory effects of unconjugated AMOs
122 on miRNA-122 (after 24 hours from co-transfection);
FIG. 7 is a graph depicting inhibitory effects of the unconjugated
AMOs 122 on miRNA-122 (after 48 hours from co-transfection);
FIG. 8 is a graph depicting inhibitory effects of the unconjugated
AMOs 122 on miRNA-122 (after 72 hours from co-transfection);
FIG. 9 is a graph depicting temporal changes in the inhibitory
effects of the unconjugated AMOs 122 on miRNA-122;
FIG. 10 is a graph depicting inhibitory effects of conjugated AMOs
122 on miRNA-122 (after 24 hours from co-transfection);
FIG. 11 is a graph depicting inhibitory effects of the conjugated
AMOs 122 on miRNA-122 (after 48 hours from co-transfection);
FIG. 12 is a graph depicting inhibitory effects of the conjugated
AMOs 122 on miRNA-122 (after 72 hours from co-transfection);
and
FIG. 13 is a graph depicting inhibitory effects of AMO122_SMe_PS on
miRNA-122 (after 48 hours from transfection) (Real-Time PCR
Method).
DESCRIPTION OF EMBODIMENTS
Embodiments of the present invention will be described in detail
below.
(1. Oligonucleotide Derivative)
First, a detailed description will be given of the structure of an
oligonucleotide derivative according to the present invention.
The oligonucleotide derivative according to the present invention
comprises repeating structural units represented by the following
general formula (1). In the present specification, the term
oligonucleotide derivative means that a nucleotide in an
oligonucleotide is chemically modified as represented by the
following general formula (1). The oligonucleotide derivative
according to the present invention also includes an oligonucleotide
derivative in which at least one nucleotide in an oligonucleotide
is chemically modified as represented by the following general
formula (1). In this case, an oligonucleotide derivative to be
preferably used is an oligonucleotide derivative in which 50% or
more of nucleotides in the entire length of the oligonucleotide are
chemically modified as represented by the following general formula
(1):
##STR00004##
In the general formula (1), B represents adenine, guanine,
cytosine, or uracil. Structures and abbreviation codes of the
individual bases are indicated in the following formulas:
##STR00005##
In the general formula (1), X represents a sulfur atom or an oxygen
atom. In the present specification, an oligonucleotide derivative
in which X represents a sulfur atom is referred to as PS, and an
oligonucleotide derivative in which X represents an oxygen atom is
referred to as PO. The PS and the PO, respectively, are represented
by respective formulas below:
##STR00006##
In the general formula (1), n represents the number of the
repeating structural units represented by the general formula (1),
namely, the number of monomers in the oligonucleotide derivative
according to the present invention, which is an integer of 6 to 60.
The number of the monomers (=n) will be described below.
In the general formula (1), B and X are independently represented
in each of the repeating structural units. In other words, in the
oligonucleotide derivative, repeating structural units having
different B and X may be present together. For example, in a single
repeating structural unit included in the oligonucleotide
derivative, B may represent adenine and X may represent a sulfur
atom, whereas, in another repeating structural unit in the same
oligonucleotide derivative, B may represent guanine and X may
represent an oxygen atom, and in still another repeating structural
unit in the same oligonucleotide derivative, B may represent
cytosine and X may represents an oxygen atom.
In the oligonucleotide derivative according to the present
invention, X represents a sulfur atom in at least one of the
repeating structural units represented by the general formula (1).
In other words, the oligonucleotide derivative according to the
present invention includes at least one repeating structural unit
represented by the general formula (1) in which X represents a
sulfur atom, namely, PS.
In the oligonucleotide derivative according to the present
invention, X may represent a sulfur atom in all of the repeating
structural units represented by the general formula (1). In other
words, all of the repeating structural units represented by the
general formula (1) included in the oligonucleotide derivative may
be PS. The oligonucleotide derivative comprising PS, namely,
phosphorothioate has excellent effect persistence enough for in
vivo use and thermal stability and therefore can be preferably used
as the oligonucleotide derivative according to the present
invention.
Next, a detailed description will be given of a function of the
oligonucleotide derivative according to the present invention.
The oligonucleotide derivative according to the present invention
can be widely used as an RNA-targeting nucleic acid (an antisense
nucleic acid compound). In the present specification, the antisense
nucleic acid compound means an oligonucleotide derivative
comprising a sequence complementary to the entire sequence or a
partial sequence of a target RNA, in which all or part of
nucleotides in oligonucleotide are chemically modified. The
oligonucleotide derivative according to the present invention can
be used, for example, for direct regulation of mRNA, regulation of
miRNA function, and the like.
When the oligonucleotide derivative according to the present
invention is used for direct regulation of mRNA, the
oligonucleotide derivative to be used is an oligonucleotide
derivative comprising a sequence complementary to the entire
sequence or a partial sequence of a target mRNA. In this case, for
example, due to a mechanism as below, translational inhibition
occurs. For example, during a splicing process from pre-mRNA to
mRNA, the oligonucleotide derivative according to the present
invention binds to the pre-mRNA to cause inhibition of cap
formation, splicing inhibition, cleavage by RNase, adenylation
inhibition, and the like. In addition, for example, during the
process of translation from mRNA, by bonding of the oligonucleotide
derivative according to the present invention to mRNA, ribosome
binding inhibition (Translational Arrest) occurs. The
oligonucleotide derivative according to the present invention has
excellent effect persistence enough for in vivo use and thermal
stability and therefore can efficiently directly regulate mRNA.
When the oligonucleotide derivative according to the present
invention is used for direct regulation of mRNA, an oligonucleotide
derivative of 6- to 60-mers is preferably used, and an
oligonucleotide derivative of 15- to 25-mers is more preferably
used. An oligonucleotide derivative having a length that allows the
advantageous effects of the present invention to be obtained can be
appropriately selected. Additionally, it should be understood that
the present invention also includes aspects of a method for
directly regulating mRNA using the oligonucleotide derivative
according to the present invention.
The oligonucleotide derivative according to the present invention
can be preferably used, for example, for regulation of miRNA
function.
In recent years, it has been pointed out that miRNA regulates
biologically importance functions such as cell proliferation and
reproductive function and has associations with various diseases.
Examples of known physiological functions regulated by miRNA
include differentiation, cell proliferation, fertility, apoptosis,
metabolism, hematopoiesis, cardiogenesis, morphogenesis, insulin
secretion, and signal transduction. Accordingly, miRNA plays an
important role in the presence of cells, and it is known that
regulation failure of gene expression due to miRNA expression
abnormalities or the like causes diseases such as cancer.
In recent years, about 900 miRNAs have been identified. Examples of
miRNAs up-regulated and down-regulated in various cancers are
listed below (Table 1).
TABLE-US-00001 TABLE 1 Organ Cancer Up-regulated Down-regulated HCA
and FNH miR-224 let-7, 122a, 422b, 203, 200c Liver HCC 21, 224,
10b, 221, 222, 20, 18 199a, 199b, 200b, 223, 122, 214, 145, 150
Cholangiocarcinomas 21, 23a, 141, 200b, 27a PET miR-23a, 342, 26a,
30d, 26b, 155, 326, 339, 326 103, 107 Insulinomas 203, 204, 211,
Pancreas PACC 23a, 342, 26a, 30d, 26b, 103, 155, 326, 229, 326 105
Ductal 21, 221, 181a, 155, 222, 181b, 148a, 375 adenocarcinomas 107
Esophagus ESCC miR-25, 424, 151 100, 99, 29c, 140, 205, 203, 202
miR-21, 223, 25, 17-5p, 125b, Stomach Adenocarcinomas 181b, 106a,
107, 92, 103, 221, let-7, 136, 218, 212, 96, 339 93, 100, 106b
Adenomas miR-21 let-7, 34, 127, 133b, 143 Colon Adenocarcinomas 21,
92, 20a, 106a, 92, 223 145 Adenocarcinomas stage II Hematopoietic
CLL miR-190, 33, 19a, 140, 123, 181b, 220 tissue 10b, 92, 188, 154,
217, 101, 196, 134, 141, 132, 192, 16, 15 Ovary Carcinomas
miR-200a, 200c let-7, 100, 101, 105, 125a, 125b, 126, 133, 140,
143, 147, 199a, 199b, 224, 9, 99a Breast Carcinomas miR-155, 21
125b, 145, 10b Lung NSCLC miR-21, 191, 155, 210 126, 224 Pituitary
gland Adenoma miR-15, 16 Prostate Carcinomas miR-32, 182, 31, 26a,
200c 520h, 494, 490, 133a, 1, 218, 220, 128a Thyroid Papillary
carcinomas miR-221, 221, 146a, 181b Anaplastic carcinomas 125b,
26a, 30a-5p Names of miRNA: miR-xx (x represents numeric values;
lin-4 and let-7 are exceptions). Visone, R. & Croce, C. Am J
Pathol. 174, 1131-1138 (2009).
For example, miRNA-21 (SEQ ID NO: 1) is up-regulated in many
cancers, such as liver cancer, pancreatic cancer, stomach cancer,
breast cancer, and lung cancer (Table 1).
For example, miRNA-122 (SEQ ID NO: 2) is a miRNA specifically
expressing in liver and is known to increase in embryo formation in
mice to regulate the development of the liver. It has also been
found that miRNA-122 is involved in the regulation of cholesterol
and lipid metabolism and the replication of HCV.
The oligonucleotide derivative according to the present invention
may comprise a sequence complementary to the entire sequence or a
partial sequence of a miRNA. In this case, the oligonucleotide
derivative can be used as a miRNA-targeting antisense nucleic acid
(anti-miRNA oligonucleotide, which is hereinafter referred to as
AMO). When an AMO according to the present invention comprises a
sequence complementary to a partial sequence of a miRNA, a sequence
allowing the present invention to be effective can be appropriately
selected as the sequence of the AMO.
When the oligonucleotide derivative according to the present
invention (hereinafter referred to as AMO according to the present
invention) comprising a sequence complementary to the entire
sequence or a partial sequence of a miRNA is administered, for
example, in vivo, the AMO according the present invention forms
double strands together with the miRNA in vivo, leading to the
inhibition of function of the miRNA. The AMO according the present
invention has excellent effect persistence enough for in vivo use
and thermal stability and therefore can efficiently regulate miRNA
function.
The AMO according the present invention can target various miRNAs,
because AMOs having sequences complementary to various miRNAs can
be synthesized. Examples of miRNAs that can be targeted by the AMO
according the present invention include miRNA-21, miRNA-122,
miRNA-224, miRNA-10b, miRNA-221, miRNA-222, miRNA-20, miRNA-18,
miRNA-23a, miRNA-141, miRNA-200b, miRNA-27a, miRNA-342, miRNA-26a,
miRNA-30d, miRNA-26b, miRNA-107, miRNA-203, miRNA-204, miRNA-211,
miRNA-105, miRNA-181a, miRNA-155, miRNA-181b, miRNA-25, miRNA-424,
miRNA-151, miRNA-223, miRNA-25, miRNA-17-5p, miRNA-125b,
miRNA-106a, miRNA-92, miRNA-103, miRNA-93, miRNA-100, miRNA-106b,
miRNA-20a, miRNA-190, miRNA-33, miRNA-19a, miRNA-140, miRNA-123,
miRNA-188, miRNA-154, miRNA-217, miRNA-101, miRNA-196, miRNA-134,
miRNA-132, miRNA-192, miRNA-16, miRNA-15, miRNA-200a, miRNA-200c,
miRNA-191, miRNA-210, miRNA-32, miRNA-182, miRNA-31, and
miRNA-146a. In addition, examples of miRNAs that can be targeted by
the AMO according to the present invention include miRNAs compiled
in a database at "miRBase: the microRNA database
(http://www.mirbase.org/)". Any of the miRNAs against which the
present invention is effective can be appropriately selected.
In the present specification, the term miRNA function means
physiological functions such as cell proliferation and regenerative
function, possessed by the various miRNAs described above. Any of
such miRNA functions against which the present invention is
effective can be appropriately selected.
The AMO according to the present invention may comprise a sequence
complementary to the entire sequence or a partial sequence of
miRNA-21 so that miRNA-21 is targeted. In this case, one example of
the AMO is an AMO having a sequence of SEQ ID NO: 3 (for example,
AMO21_SMe_PS). A formula of repeating structural units of
AMO21_SMe_PS is indicated below:
##STR00007##
The AMO according to the present invention may comprise a sequence
complementary to the entire sequence or a partial sequence of
miRNA-122 so that miRNA-122 is targeted. In this case, one example
of the AMO is an AMO having a sequence of SEQ ID NO: 5 (for
example, AMO122_SMe_PS). A formula of repeating structural units of
the AMO122_SMe_PS is indicated below:
##STR00008##
When the oligonucleotide derivative according to the present
invention is used as an AMO, an AMO of 6- to 60-mers is preferably
used, an AMO of 10- to 40-mers is more preferably used, and an AMO
of 15- to 25-mers is still more preferably used. An AMO having a
length of an oligonucleotide derivative allowing the present
invention to be effective can be appropriately selected.
In the AMO according to the present invention, an additional
oligonucleotide derivative of 1- to 20-mers (hereinafter referred
to as an additional sequence) may be bound to the 5' end, the 3'
end, or both the 5' end and the 3' end of an olignucleotide
derivative comprising a sequence complementary to the entire
sequence or a partial sequence of a target miRNA. For example, in
an miRNA-21-targeting AMO having a sequence of SEQ ID NO: 4
(32-mers), an additional sequence of 5-mers is bound to each of the
5' end and the 3' end of AMO21_SMe_PS (22-mers). Any additional
sequence allowing the present invention to be effective can be
appropriately selected.
In addition, it should be understood that the present invention
also includes aspects of a method for regulating miRNA function
using the oligonucleotide derivative according to the present
invention.
Evaluation of the inhibition of miRNA function by the
oligonucleotide derivative according to the present invention can
be performed, for example, as follows: when targeting miRNA-122,
the oligonucleotide derivative according to the present invention
is transfected into cells and then the level of miRNA-122 in the
cells is quantified to confirm that the level of the miRNA-122 is
lower than that in untreated cells. Alternatively, the evaluation
can be performed, for example, by administering the oligonucleotide
derivative according to the present invention in a mammal and
quantifying the level of miRNA-122 in the liver to confirm that the
miRNA-122 level is lower than before the administration
thereof.
Next, a detailed description will be given of a conjugated
oligonucleotide derivative according to the present invention.
The oligonucleotide derivative according to the present invention
may be a conjugated oligonucleotide derivative having at least one
ligand bound thereto. In the present specification, the term ligand
means a substance that allows cell targeting, tissue targeting,
functionality improvement, and the like by the oligonucleotide
derivative according to the present invention.
In the conjugated oligonucleotide derivative, a ligand can be bound
to the 5' end, the 3' end, or both the 5' end and the 3' end of an
oligonucleotide derivative. The ligand can be bound to the
oligonucleotide derivative by an usual method.
In the conjugated oligonucleotide derivative, a plurality of
ligands may be bound to an oligonucleotide derivative. In this
case, a conjugated oligonucleotide derivative having two to five
ligands bound thereto is preferably used, and a conjugated
oligonucleotide derivative having three ligands bound thereto is
more preferably used. When three ligands are bound thereto, for
example, the three ligands may be bound to the 5' end or the 3' end
of the oligonucleotide derivative. Alternatively, for example, one
of the three ligands may be bound to the 5' end thereof and two of
the three ligands may be bound to the 3' end thereof. The number of
ligands can be appropriately selected in a range allowing the
present invention to be effective.
Examples of ligands usable in the conjugated oligonucleotide
derivative according to the present invention include tocopherol,
cholesterol, PSMA (prostate-specific membrane antigen),
polyethylene glycol, vitamin A, folic acid, fatty chain, peptides,
transferrins, aptamers, mannose, GalNAC(N-acetylgalactosamine),
anisamide, other surface antigen-recognizing low molecular weight
compounds or high molecular weight compounds, and combinations
thereof. In a conjugated oligonucleotide derivative having a
plurality of ligands bound thereto, the ligands bound thereto may
be a plurality of ligands of the same kind or a combination of
ligands of different kinds. Any ligand that allows the effects of
the present invention to be obtained can be appropriately
selected.
Examples of the conjugated oligonucleotide derivative according the
present invention include conjugated AMOs targeting miRNA-122 as
below:
AMO122_SMe_PS_5'Toc (SEQ ID NO: 5): tocopherol is bound to the 5'
end of AMO122_SMe_PS;
AMO122_SMe_PS_5'Chol (SEQ ID NO: 5): cholesterol is bound to the 5'
end of AMO122_SMe_PS;
AMO122_SMe_PS_3'Chol (SEQ ID NO: 5): cholesterol is bound to the 3'
end of AMO122_SMe_PS; and
AMO21_SMe_PS_5'PMSA (SEQ ID NO: 3): PMSA (prostate membrane
antigen) is bound to the 5' end of AMO21_SMe_PS.
##STR00009##
Conjugated oligonucleotide derivatives according to the present
invention allow cell targeting, tissue targeting, functionality
improvement, and the like by oligonucleotide derivative. For
example, a conjugated oligonucleotide derivative using tocopherol
as a ligand can achieve liver tissue targeting, and a conjugated
oligonucleotide derivative using PSMA as a ligand can achieve
prostate tissue targeting. For example, a conjugated
oligonucleotide derivative using polyethylene glycol as a ligand
improves retainability in blood. In addition, in a conjugated
oligonucleotide derivative having a plurality of ligands bound
thereto, for example, binding of a plurality of ligands that will
bind to a certain kind of receptor can enhance interaction between
the ligands and the receptor, further ensuring tissue targeting.
Furthermore, for example, by using a conjugated oligonucleotide
derivative having a combination of tocopherol and polyethylene
glycol, as a ligand, bound thereto, tissue targeting and
retainability in blood can be achieved simultaneously, thereby
further ensuring liver tissue targeting.
Next, a description will be given of a method for synthesizing the
oligonucleotide derivative according to the present invention.
The oligonucleotide derivative according to the present invention
can be synthesized by an amidite method using a DNA synthesizer.
For example, an AMO comprising SMe_PS (phosphorothioate) can be
synthesized using a controlled pore glass (CPG) on which a
2'-OMe-4'-thio (in which a hydroxyl group at position 2' of a
furanose ring of the sugar moiety of a nucleic acid has been
methylated and an oxygen atom at position 4' of the furanose ring
of the sugar moiety thereof has been substituted with a sulfur
atom) ribonucleoside is supported and an amidite of 2'-OMe-4'-thio
ribonucleoside, by sulfurization of phosphoric acid with
3H-1,2-benzodithiol-3-one-1,1-dioxide (Beaucage reagent). As an
conjugated oligonucleotide derivative, for example,
AMO122_SMe_PS_5'Toc can be synthesized in the same manner as above
using an .alpha.-tocopherol amidite synthesized from
.alpha.-tocopherol, and for example, AMO21_SMe_PS_5'PMSA can be
synthesized in the same manner as above using an amidite of a
5'PMSA ligand synthesized from L-lysine. Various CPGs and amidites
used in condensation reaction by the amidite method may be
commercially available products. Any synthesis method that allows
the effects of the present invention to be obtained can be
appropriately selected.
(2. Pharmaceutical Composition for Treatment)
A pharmaceutical composition for treatment including the
oligonucleotide derivative according to the present invention
exhibits treatment effect, for example, through direct regulation
of mRNA, regulation of miRNA function, or the like.
Regarding treatment effect through the regulation of miRNA
function, for example, since miRNA-21 is up-regulated in various
cancers such as liver cancer, pancreatic cancer, stomach cancer,
breast cancer, and lung cancer as described above, administration
of an miRNA-21-targeting AMO can inhibit the function of miRNA-21
in vivo and thus can exhibit treatment effects against the above
cancers. In addition, for example, since miRNA-122 is involved in
the replication of HCV as described above, administration of a
miRNA-122-targeting AMO can inhibit the function of miRNA-122 and
thus can exhibit a treatment effect against hepatitis C. Any
disease against which the present invention can be effectively
applied can be appropriately selected.
The oligonucleotide derivative according to the present invention
has excellent effect persistence enough for in vivo use and thermal
stability. Therefore, efficient regulation of miRNA function can be
achieved and thus effective treatment effect is obtainable.
Administration of the pharmaceutical composition for treatment
according the present invention to mammals can be performed, for
example, through injection, oral administration, sublingual
administration, and the like. Examples of injection administration
include intravenous administration, intraarterial injection,
intradermal injection, subcutaneous injection, intramuscular
injection, and intraperitoneal injection. In addition, regarding
dosage form, the pharmaceutical composition can be appropriately
prepared into injection, sublingual tablet, granules, powder, or
the like. For example, when the pharmaceutical composition is an
injection, the composition can be prepared into an aqueous
injection, a nonaqueous injection, a suspension injection, a solid
injection, or the like, for an injection agent. When the
composition is prepared into an injection agent, one or more
additives may also be added, such as a solubilizer, a buffering
agent, a tonicity agent, a stabilizer, a preservative, and/or a
soothing agent. When prepared as an oral agent, an additives, a
binder, a disintegrating agent, a thickener, and/or a dispersing
agent, or the like that are commonly used can be appropriately
included. The pharmaceutical composition for treatment may further
appropriately include other active ingredients. Any administration
method, any dosage form, any additive, and the like that allow the
effects of the present invention to be obtained can be
appropriately selected.
When the pharmaceutical composition for treatment according to the
present invention is administered to a mammal, for example,
administration may be carried out by dissolving the oligonucleotide
derivative in a solvent commonly used for injection agents or
dissolving the oligonucleotide derivative embedded in liposome in a
solvent. Any administration method that allows the effects of the
present invention to be obtained can be appropriately selected.
(3. Pharmaceutical Composition for Diagnosis)
A pharmaceutical composition for diagnosis including the
oligonucleotide derivative according to the present invention
exhibits treatment effect, for example, through the regulation of
miRNA function.
For example, when carrying out in-vivo administration of an AMO
targeting an miRNA as a biomarker of a specific cancer, a tracer
detectable from outside the body is bound to the AMO in advance,
with the result that binding of the AMO to the target miRNA enables
the diagnosis of cancer by means of imaging (for example, PET). In
addition, for example, by adding an AMO labelled with some label
(such as a fluorescent label or a radioisotope label) into a
tissue, blood, or the like collected from a living body, the
confirmation of intracellular expression of the target miRNA and
functional analysis thereof can be performed, as well as the
quantification of the target miRNA in the tissue, the blood, or the
like can also be performed. In addition to that, for example, by
administering the AMO in vivo to inhibit the expression of the
target miRNA, miRNA expression analysis can be made. Any diagnostic
use for which the present invention is effective can be
appropriately selected.
The AMO according to the present invention has excellent effect
persistence enough for in vivo use and thermal stability and
therefore can stably bind to a target miRNA in vivo and can inhibit
the function of the target miRNA, thereby ensuring diagnosis using
the AMO.
The administration method, the dosage form, additives, and the like
for the pharmaceutical composition for diagnosis according to the
present invention are the same as those described above.
(4. Oligonucleotide Derivative for Inhibiting miRNA Function)
An oligonucleotide derivative for inhibiting miRNA function
according to the present invention comprises, similarly as
described above, repeating structural units represented by the
following general formula (1):
##STR00010## (wherein B represents adenine, guanine, cytosine, or
uracil; X represents a sulfur atom or an oxygen atom; n represents
an integer of 6 to 60; and B and X are independently represented in
each of the repeating structural units), wherein X represents a
sulfur atom in at least one of the repeating structural units
represented by the general formula (1). In addition, in the
oligonucleotide derivative for inhibiting miRNA function according
to the present invention, similarly as described above, X may
represent a sulfur atom in all of the repeating structural units
represented by the general formula (1). Such an oligonucleotide
derivative for inhibiting miRNA function comprising PS, namely,
phosphorothioate has excellent effect persistence enough for in
vivo use and thermal stability and therefore can be preferably used
in the present invention.
The oligonucleotide derivative for inhibiting miRNA function
according to the present invention may have, similarly to the
above, at least one ligand bound to the 5' end, the 3' end, or both
the 5' end and the 3' end. Such a conjugated oligonucleotide
derivative for inhibiting miRNA function can achieve cell
targeting, tissue targeting, functionality improvement, and the
like by oligonucleotide derivative.
The oligonucleotide derivative for inhibiting miRNA function
according to the present invention can inhibit miRNA function in
vivo or in vitro. The oligonucleotide derivative for inhibiting
miRNA function according to the present invention can target
various miRNAs, similarly to the above.
Evaluation of the inhibition of miRNA function by the
oligonucleotide derivative for inhibiting miRNA function according
to the present invention can be performed as follows. In the same
way as above, for example, when miRNA-122 is targeted, an
oligonucleotide derivative for inhibiting miRNA-122 function
according to the present invention is transfected into cells, then,
the level of miRNA-122 in the cells is quantified to confirm that
the level of miRNA-122 is less than that in the untreated cells. In
addition, for example, the evaluation can be performed by
administering the oligonucleotide derivative for inhibiting
miRNA-122 function according to the present invention to a mammal
and quantifying the level of miRNA-122 in the liver to confirm that
the level of miRNA-122 after the administration is lower than
before the administration.
The oligonucleotide derivative for inhibiting miRNA function
according to the present invention may comprise, similarly to the
above, a sequence complementary to the entire sequence or a partial
sequence of miRNA. In this case, the derivative can be used as a
miRNA-targeting AMO. When the oligonucleotide derivative for
inhibiting miRNA function according to the present invention is
administered, for example, in vivo, the oligonucleotide derivative
forms double strands together with the miRNA in vivo, thereby
inhibiting miRNA function. Additionally, the miRNA in this case may
be, similarly to the above, miRNA-21 or miRNA-122.
The present invention is not limited to the embodiments described
above and various modifications and applications can be made.
Example 1
Hereinbelow, a detailed description will be given of the present
invention with reference to Examples. However, the present
invention is not limited to the Examples. In addition, "%"
represents % by mass unless otherwise specified.
(Synthesis of AMOs)
Each AMO was synthesized in the following manner. The name and SEQ
ID NO of each AMO are indicated below. In the following Examples,
an AMO that is fully complementary to miRNA-21 is referred to as
AMO 21, and an AMO that is fully complementary to miRNA-122 is
referred to as AMO 122.
1. Unconjugated AMOs 21
AMO21(32)_SMe (SEQ ID NO: 4)
AMO21_SMe_PO (SEQ ID NO: 3)
AMO21_SMe_PS (SEQ ID NO: 3)
2. Unconjugated AMOs 122
AMO122_SMe_PO (SEQ ID NO: 5)
AMO122_SMe_PS (SEQ ID NO: 5)
3. Conjugated AMOs 122
AMO122_SMe_PS_5'Toc (SEQ ID NO: 5) AMO122_SMe_PS_5'Chol (SEQ ID NO:
5)
AMO122_SMe_PS_3'Chol (SEQ ID NO: 5)
4. Conjugated AMOs 21
AMO21_SMe_PS_5'PMSA (SEQ ID NO: 3)
5. Comparative Examples
Unconjugated AMOs 21
AMO21(32)_Me (SEQ ID NO: 4)
AMO21_Me_PO (SEQ ID NO: 3)
AMO21_FMe_PO (a methoxy group in the 2' position of AMO21_Me_PO is
substituted with fluorine) (SEQ ID NO: 3)
AMO21_SFMe_PO (a methoxy group in the 2' position of AMO21_SMe_PO
is substituted with fluorine) (SEQ ID NO: 3)
6. Comparative Examples
Unconjugated AMOs 122
AMO122_Me_PO (SEQ ID NO: 5)
AMO122_Me_PS (SEQ ID NO: 5)
7. Comparative Examples
Conjugated AMOs 122
AMO122_Me_PS_5'Toc (SEQ ID NO: 5)
AMO122_Me_PS_5'Chol (SEQ ID NO: 5)
AMO122_Me_PS_3'Chol (SEQ ID NO: 5)
8. Comparative Example
Conjugated AMO 21
AMO21_Me_PS_5'PMSA (SEQ ID NO: 3)
The following formulas represent repeating structural units of the
unconjugated AMOs 21:
##STR00011##
The following formulas represent repeating structural units of the
unconjugated AMOs 122:
##STR00012##
The following formulas represent structures and repeating
structural units of the conjugated AMOs 122 and the conjugated AMO
21.
##STR00013##
The following formulas represent repeating structural units of the
unconjugated AMOs 21 used in the Comparative Examples:
##STR00014##
The following formulas represent repeating structural units of the
unconjugated AMO122 used in the Comparative Examples:
##STR00015##
The following formulas represent structures and repeating
structural units of the conjugated AMO122 and the conjugated AMO21
used in the Comparative Examples:
##STR00016##
(A. Methods for Synthesizing AMO Comprising Me_PO and AMO
Comprising SMe_PO)
The AMOs were synthesized using a DNA synthesizer: ABI 3400
(manufactured by Applied Biosystem Co., Ltd.) in accordance with a
usual DNA solid phase synthesis method.
Condensation reaction of the AMO comprising Me_PO was performed
using a CPG on which 1 .mu.mol of 2'-OMe (a hydroxyl group at
position 2' of a furanose ring of the sugar moiety of a nucleic
acid has been methylated) nucleoside is supported (manufactured by
Glen Research Co., Ltd.) and a 2'-OMe nucleoside amidite
(manufactured by Glen Research Co., Ltd). The 2'-OMe nucleoside
amidite was prepared in a 0.1 M acetonitrile solution to be used
for condensation reaction.
Condensation reaction of the AMO comprising SMe_PO was performed
using a CPG on which 1 .mu.mol of 2'-OMe-4'-thio (a hydroxyl group
at position 2' of a furanose ring of the sugar moiety of a nucleic
acid has been methylated and an oxygen atom at position 4' of the
sugar moiety furanose ring has been substituted with a sulfur atom)
ribonucleoside is supported and a 2'-OMe-4'-thio ribonucleoside
amidite. The CPG with the 2'-OMe-4'-thio ribonucleoside supported
thereon and the 2'-OMe-4'-thio ribonucleoside amidite,
respectively, were synthesized from the CPG with the 2'-OMe
nucleoside supported thereon and the 2'-OMe nucleoside amidite,
respectively, in accordance with a usual method. The 2'-OMe-4'-thio
ribonucleoside amidite was prepared in a 0.1 M acetonitrile
solution to be used for condensation reaction.
By adding 3% TCA (trichloroacetic acid), a DMTr group in the CPG
with the 2'-OMe nucleoside supported thereon or the CPG with the
2'-OMe-4'-thio ribonucleoside supported thereon was removed, and
then, 1H-tetrazole was used as an activator to perform the
condensation of the CPGs, respectively, with the 2'-OMe nucleoside
amidite or the 2'-OMe-4'-thio ribonucleoside amidite, respectively.
Condensation time was 600 seconds. Next, after capping by reacting
acetic anhydride with unreacted hydroxyl group, oxidation of
phosphoric acid was performed using iodine as an oxidizer in the
presence of water. This cycle was repeated to synthesize an AMO
supported on the solid phase (CPG). The following represents a
synthesis scheme for the AMO comprising SMe_PO.
Synthesis Scheme for AMO Comprising SMe_PO
##STR00017##
(B. Methods for Synthesizing AMO Comprising Me_PS and AMO
comprising SMe_PS)
Instead of iodine as the oxidizer in section A above,
3H1,2-benzodithiol-3-one-1,1-dioxide (Beaucage reagent) was used
for the sulfurization of phosphoric acid to synthesize AMOs in the
same manner as section A above. The following is a synthesis scheme
for the AMO comprising SMe_PS.
Synthesis Scheme for AMO Comprising SMe_PS
##STR00018##
(C. Method for Synthesizing Conjugated AMO)
An .alpha.-tocopherol amidite was prepared in 0.1 M of a 10%
THF/acetonitrile solution and, condensation reactions were
performed in the same manner as section B above to obtain
AMO122_Me_PS_5'Toc and AMO122_SMe_PS_5'Toc. The .alpha.-tocopherol
amidite was synthesized from .alpha.-tocopherol as below:
##STR00019##
A 5'-Cholesteryl-TEG (triethylene glycol) amidite (manufactured by
Glen Research Co., Ltd.) was prepared in 0.1 M of a 10%
THF/acetonitrile solution, and condensation reactions were
performed in the same manner as section B above to obtain
AMO122_Me_PS_5'Chol and AMO122_SMe_PS_5'Chol.
Using a CPG resin with 1 .mu.mol of 3'-Cholesteryl-TEG supported
thereon (manufactured by Glen Research Co. Ltd.), condensation
reactions were performed in the same manner as section B above to
obtain AMO122_Me_PS_3'Chol and AMO122_SMe_PS_3'Chol.
A 5'-PMSA ligand amidite was prepared in 0.1 M of acetonitrile
solution, and condensation reactions were performed in the same
manner as section B above to obtain AMO21_Me_PS_5'PMSA and AMO21
SMe_PS_5'PMSA. The 5'-PMSA ligand amidite was synthesized from
L-lysine as below:
##STR00020##
(D. Methods for Synthesizing AMO21_FMe_PO and AMO21_SFMe_PO)
The Comparative Examples AMO21_FMe_PO and AMO21_SFMe_PO were
synthesized in the same manner as section A above in accordance
with the usual method.
After completion of the syntheses, each CPG with each AMO supported
thereon was transferred into a vial. Then, 28% ammonia
water/ethanol (3:1, 2 mL) was added and each vial was allowed to
stand at 55.degree. C. for 16 hours to perform cleavage and
deprotection of the each AMO. The reaction solution was filtered
with a glass filter and the solvent was distilled away under
reduced pressure. The each unconjugated AMO synthesized in the
state of having a DMTr group left at the 5' end thereof was roughly
purified by C18 reverse phase HPLC (J' sphere YMC ODS-M80,
150.times.4.6 mm, 5 to 50% acetonitrile in 0.1 N TEAA buffer, pH
7.0) to collect a fraction containing a full-length AMO having the
DMTr group, and then the solvent was distilled away under reduced
pressure. After performing desalination of the residue using
Sep-Pak C18 (Waters), hydrochloric acid (pH 2.0) was added and
treated at room temperature for 20 minutes to remove the DMTr group
left at the 5' end. The obtained reaction solution was neutralized
with diluted ammonia aqueous solution and then the solvent was
distilled away under reduced pressure. The residue containing each
of the conjugated and the unconjugated full-length AMOs was
purified by C18 reverse phase HPLC (J' sphere YMC ODS-M80,
150.times.4.6 mm, 5 to 50% acetonitrile in 0.1 N TEAA buffer, pH
7.0), and then, desalination was performed using Sep-Pak C18 to
obtain each AMO with high purity.
The structure of each AMO purified was analyzed by MALDI-TOF/MASS
spectrometry (ULTRAFLEX TOF/TOF, manufactured by Bruker Daltonics)
to obtain a molecular weight thereof. The analysis results are
listed below:
AMO21_Me_PO: calculated mass,
C.sub.231H.sub.302N.sub.82O.sub.150P.sub.21 7276.3 (M-H); observed
mass, 7273.80.
AMO21_SMe_PO: calculated mass,
C.sub.231H.sub.302N.sub.82O.sub.128P.sub.21S.sub.22 7627.80 (M-H);
observed mass, 7626.50.
AMO122_Me_PO: calculated mass,
C.sub.240H.sub.318N.sub.85O.sub.154P.sub.22 7539.40 (M-H); observed
mass, 7538.52.
AMO122_Me_PS: calculated mass,
C.sub.240H.sub.318N.sub.85O.sub.132P.sub.22S.sub.22 7891.89 (M-H);
observed mass, 7886.64.
AMO122_SMe_PO: calculated mass,
C.sub.240H.sub.318N.sub.85O.sub.131P.sub.22S.sub.23 7906.87 (M-H);
observed mass, 7906.72.
AMO122_SMe_PS: calculated mass,
C.sub.240H.sub.318N.sub.85O.sub.109P.sub.22S.sub.45 8261.36 (M-H);
observed mass, 8261.66.
AMO122_Me_PS_5'Toc: calculated mass,
C.sub.275H.sub.378N.sub.86O.sub.138P.sub.23S.sub.23 8543.28 (M-H);
observed mass, 4546.78.
AMO122_SMe_PS_5'Toc: calculated mass,
C.sub.275H.sub.378N.sub.86O.sub.115P.sub.23S.sub.46 8913.75 (M-H);
observed mass, 8916.81.
AMO122_Me_PS_5'Chol: calculated mass,
C.sub.281H.sub.390N.sub.86O.sub.140P.sub.23S.sub.23 8661.36 (M-H);
observed mass, 8662.17.
AMO122_SMe_PS_5'Chol: calculated mass,
C.sub.281H.sub.390N.sub.86O.sub.112P.sub.23S.sub.46 9030.83 (M-H);
observed mass, 9033.77.
AMO122_Me_PS_3'Chol: calculated mass,
C.sub.281H3.sub.90N.sub.86O.sub.140P.sub.23S.sub.23 8661.36 (M-H);
observed mass, 8662.04.
AMO122_SMe_PS_3'Chol: calculated mass,
C.sub.281H.sub.390N.sub.86O.sub.112P.sub.23S.sub.46 9030.83 (M-H);
observed mass, 9032.13.
AMO122_Me_PS_5'PSMA: calculated mass,
C.sub.252H.sub.338N.sub.85O.sub.143P.sub.22S.sub.22 8231.99 (M-H);
observed mass, 8231.99.
AMO122_SMe_PS_5'PSMA: calculated mass,
C.sub.252H.sub.338N.sub.85O.sub.121P.sub.22S.sub.44 8585.49 (M-H);
observed mass, 8585.49.
FIG. 1 indicates sequences and 50% melting temperatures (Tm values)
of the unconjugated AMO21 (22-mer series: SEQ ID NO: 3; 32-mer
series: SEQ ID NO: 4). The results of measurement of the Tm values
(measurement conditions: 10 mM of phosphate buffer (pH 7.0), 0.1 mM
of EDTA, 1 mM of sodium chloride, 3 .mu.M of strand concentration)
showed that the unconjugated AMOs 21 according to the Example of
the present invention has an ability to form thermally stable
double strands together with miRNA-21 as complementary chain
RNA.
FIG. 2 indicates sequences and Tm values of the unconjugated AMO122
(SEQ ID NO: 5). The results of measurement of the Tm values
(measurement conditions were the same as those described above)
showed that the unconjugated AMOs 122 according to the Example of
the present invention have an ability to form thermally stable
double strands together with miRNA-122 as complementary chain
RNA.
Example 2
Construction of miRNA Reporter Vector
A sequence formed by arranging two fully complementary sequences of
miRNA-21 (SEQ ID NO: 6) or two fully complementary sequences of
miRNA-122 (SEQ ID NO: 7) in series was cloned in a 3' untranslated
region (3'-UTR) of a firefly luciferase gene in a pmirGLO vector
(purchased from Promega K.K) to construct an miRNA reporter vector
(FIG. 3). The pmirGLO vector expresses firefly luciferase (Flue)
and Renilla luciferase (Rluc).
Example 3
Assay of AMOs
As indicated in FIG. 4, HeLa cells (human cervical cancer cells)
for miRNA-21 or Huh-7 cells (cells highly expressing miRNA-122) for
miRNA-122 were seeded at a density of 10,000 cells in each well of
a 96-well plate (a LumiNunc 96-well microplate) and then incubated
at 37.degree. C. in 5% CO.sub.2 in air. Twenty-four hours later,
each AMO and the above-mentioned reporter vector (0.1 .mu.g/well)
were co-transfected using LIPOFECTAMINE 2000 (purchased from
Invitrogen, Inc.) and incubated. At that time, Opti-MEM (purchased
from Invitrogen, Inc) was used as culture medium. Six hours later,
the medium was replaced by a complete medium (a DMEM containing 10%
FBS and an antibiotic) and again, incubation was performed. After
24 hours, 48 hours, and 72 hours from the co-transfection, the
cells were dissolved in Lysis buffer to measure luciferase activity
using a dual-luciferase reporter assay system (purchased from
Promega K.K). Fluc/Rluc relative ratio (%) (FIGS. 5 to 12)
represents relative values of activities of the individual
AMO-administered cells when firefly luciferase activity value is
normalized by Renilla luciferase activity and mirGLO-administered
cell activity is set as 100%. The relative values were obtained by
performing individual experiments at least three times to calculate
average values and standard deviations thereof.
(Evaluation of Activities of Unconjugated AMOs 21)
FIG. 5 indicates results obtained after 24 hours from the
co-transfection. The mirGLO represents a control in which only a
reporter vector was added. The mirGLO miR21Scr (scramble)
represents a control in which only a reporter vector having a
different 3'-UTR sequence was added. Since miRNA-21 cannot bind to
the 3'-UTR, the Fluc/Rluc relative ratio becomes high. The mirGLO
miR21 represents a control in which the reporter vector and
miRNA-21 were added. Since miRNA-21 binds to the 3'-UTR, the
Fluc/Rluc relative ratio becomes low. In addition, in FIG. 5, the
concentrations of the individual AMOs are 50 nM, 5 nM, and 0.5 nM
in order from the left of the three columns of the individual
AMOs.
As compared to the Comparative Examples AMO21(32)_Me, AMO21_Me_PO,
AMO21_FMe_PO, and AMO21_SFMe_PO, the Fluc/Rluc relative ratio was
shown to be dose-dependently higher in AMO21(32)_SMe, AMO21_SMePO,
and AMO21_SMe_PS. This is due to the fact that, by binding of the
unconjugated AMOs 21 according to the Example of the present
invention to miRNA-21, miRNA-21 was not able to bind to the 3'-UTR
of the reporter vector and therefore the Fluc/Rluc relative ratio
became high. The results showed that the AMOs 21 according to the
Example of the present invention inhibit miRNA-21.
(Evaluation of Activities of Unconjugated AMOs 122)
FIG. 6 indicates results after 24 hours from the co-transfection,
FIG. 7 indicates results after 48 hours therefrom, and FIG. 8
indicates results after 72 hours therefrom. In FIGS. 6 to 8, the
concentrations of the individual AMOs are 10 nM, 5 nM, 1 nM, and
0.5 nM in order from the left of the four columns of the individual
AMOs.
In FIGS. 6 to 8, as compared to the Comparative Examples:
AMO122_Me_PO and AMO122_Me_PS, the Fluc/Rluc relative ratio was
shown to be dose-dependently higher in AMO122_SMe_PO and
AMO122_SMe_PS.
Additionally, FIG. 9 indicates changes with the passage of time in
the activities of the unconjugated AMOs 122 at an AMO concentration
of 5 nM. As compared to the Comparative Examples AMO122_Me_PO and
AMO122_Me_PS, AMO122 activity was favorably maintained in
AMO122_SMe_PO and AMO122_SMe_PS and the AMO122 activity of the
AMO122_SMe_PS improved with the passage of time. The results showed
that the AMOs 122 according to the Example of the present invention
consistently inhibit miRNA-122.
(Evaluation of Activities of Conjugated AMOs 122)
FIG. 10 indicates results obtained after 24 hours from the
co-transfection; FIG. 11 indicates results obtained after 48 hours
therefrom; and FIG. 12 indicates results obtained after 72 hours
therefrom. In FIGS. 10 to 12, the concentrations of the individual
AMOs are 10 nM, 5 nM, 1 nM, and 0.5 nM in order from the left of
the four columns of the individual AMOs.
In FIGS. 10 to 12, as compared to the Comparative Examples
AMO122_Me_PS_5'Toc, AMO122_Me_PS_5' Chol, and AMO122_Me_PS_3'Chol,
the Fluc/Rluc relative ratio was shown to be dose-dependently
higher in AMO122_SMe_PS_5'Toc, AMO122_SMe_PS_5'Chol, and
AMO122_SMe_PS_3'Chol, where the relative ratios thereof in the AMOs
were approximately at the same level as that in the unconjugated
AMO122_SMe_PS. The results showed that the conjugated AMOs
according to the Example of the present invention inhibit miRNA, as
with the unconjugated AMOs.
Example 4
Analysis by Real-Time PCR Method
AMO122_SMe_PS or AMO122_Me_PS as Comparative Example, respectively,
was transfected in Huh-7 cells and then, the expression level of
miRNA-122 after 48 hours from the transfection was quantified by a
real-time PCR method.
The procedures of the real-time PCR will be described below.
The Huh-7 cells were cultured in a Dulbecco's modified Eagle's
medium (DMEM) (Gibco, Inc) (containing 10% fetal bovine serum
(FBS), 100 units/mL.sup.-1 penicillin, and 100 .mu.g/mL.sup.-1
streptomycin) at 37.degree. C. in 5% CO.sub.2 in air.
In DMEM (Sigma, Inc) (containing 10% FBS (Thermo Fisher Scientific,
Inc), 100 units/mL.sup.-1 penicillin, and 100 .mu.g/mL.sup.-1
streptomycin), the cells were seeded at a density of
1.5.times.10.sup.5 cells in each well of a 6-well plate.
Twenty-four hours later, AMO122_SMe_PS or AMO122_Me_PS was
transfected using LIPOFECTAMINE 2000 (purchased from Invitrogen,
Inc.) in accordance with an attached instruction manual and
incubated at 37.degree. C. At that time, Opti-MEM (purchased from
Invitrogen, Inc) was used as culture medium. After 6 hours from the
transfection, the medium was replaced by a complete medium (DMEM as
mentioned above), and again incubation was performed at 37.degree.
C. After 48 hours from the transfection, the cells were washed with
PBS and then QIAzol Lysis Reagent (Qiagen, Inc) was added to
dissolve the cells. The obtained solution was purified using
miRNeasy Mini Kit (Qiagen, Inc.) to extract total RNA.
Using 10 ng of the extracted RNA sample, reverse transcription
reaction was performed. The reverse transcription reaction was
performed using miRNA-122 specific RT primers of TaqMan (registered
trade mark) Small RNA Assays (Applied biosystems, Inc) and a TaqMan
(registered trademark) MicroRNA Reverse Transcription Kit (Applied
Biosystems, Inc.) (at 16.degree. C. for 30 minutes, at 42.degree.
C. for 30 minutes, and at 85.degree. C. for 5 minutes).
An amount of 1.33 .mu.l, of the reverse transcription reaction
solution obtained above, 1.0 .mu.L of TaqMan (registered trade
mark) Small RNA Assays (a mixture of an miRNA-122 specific forward
primer, an miRNA-122 specific reverse primer, and an miRNA-122
specific TaqMan (registered trademark) MGB probe) (Applied
biosystems, Inc), and 10 .mu.l, of TaqMan Universal PCR Master Mix
II (Applied biosystems, Inc) were mixed together to measure the
expression level of miRNA-122 in the cells by a real-time PCR
method. The real-time PCR used LightCycler (registered trademark)
480 Real-Time PCR System (Roche Applied Science, Inc) was used to
perform one cycle at 50.degree. C. for 2 minutes, one cycle at
95.degree. C. for 10 minutes, and 40 cycles at 95.degree. C. for 15
seconds and 60.degree. C. for 1 minute. At that time, the
expression level of RNU6B as an internal standard gene was used to
standardize the expression level of miRNA-122.
FIG. 13 indicates the results. In FIG. 13, when the expression
level of miRNA-122 in the "NT" (non-treated: non-treated cells) is
set as 100, the expression levels of miRNA 122 in the cells are
indicated by the proportion of expression level of miRNA-122 in
each AMO with respect to the "NT". In addition, the concentrations
of the each AMO are 5 nM and 25 nM in order from the left of the
two columns of the each AMO.
As compared to the Comparative Example AMO122_Me_PS, the expression
level of miRNA-122 in AMO122_SMe_PS was inhibited at lower levels.
In addition, the inhibition of miRNA expression level was
dose-dependent. Accordingly, the quantification by the real-time
PCR method also showed that the AMO 122 according to the Example of
the present invention inhibits miRNA-122.
As described hereinabove, the oligonucleotide derivative according
to the present invention has excellent effect persistence durable
in use in vivo and thermal stability and therefore can efficiently
regulate miRNA function.
Various embodiments and modifications are available to the present
invention without departing from the broad sense of spirit and
scope of the invention. In addition, the embodiments described
above are merely illustrative of the present invention and not to
be construed as limiting the invention. In other words, the scope
of the present invention is set forth by the appended claims, not
by the embodiments, and various modifications that come within the
scope of the claims and the scope of significance of the invention
equivalent thereto are considered to be within the scope of the
invention.
The present invention is based on Japanese Patent Application No.
2011-125734 filed on Jun. 3, 2011, and the entire specification,
scope of claims, and drawings of which are incorporated herein by
reference.
SEQUENCE LISTINGS
1
7122RNAHomo sapiens 1uagcuuauca gacugauguu ga 22223RNAHomo sapiens
2uggaguguga caaugguguu ugu 23322RNAArtificialAntisense RNA
3ucaacaucag ucugauaagc ua 22432RNAArtificialAntisense RNA
4ucuuaucaac aucagucuga uaagcuaacc uu 32523RNAArtificialAntisense
RNA 5acaaacacca uugucacacu cca 23622DNAArtificialSynthetic
oliogonucleotide complementary to miRNA-21 6tcaacatcag tctgataagc
ta 22723DNAArtificialSynthetic oliogonucleotide complementary to
miRNA-122 7acaaacacca ttgtcacact cca 23
* * * * *
References